Development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids

Abstract

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive "sample-to-result" diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

Figure 1

Rheonix processor platform (controller) and CARD technologies. (A) Controller box with integrated vacuum and pressure pump system. A manifold forms the interface between the controller box and the CARD; in the magnified top view the heating module and the solenoid connections (ports) are indicated. Vacuum and pressure ballast tanks are integrated within the controller box. (B (a)) panel showing a minimal schematic of the basic 3-layer PS CARD structure with 2 small and one larger diaphragm (valves/pumps); (B (b)) a top down view of the 3-layer laminated structure shown in panel (B (a)) (without any reservoirs mounted). (C) Diagram showing the valve operations required for peristaltic fluid movement in the CARD.

Figure 2

The dual-path antibody RNA CARD. Design (a) and top down image (b) of the dual path microfluidic device with the reagent reservoirs and other compartments used in the detection of anti-HIV antibody and HIV RNA.

Figure 3

Unique modular components of the CARD. (A) Saliva collector with removable solid Porex matrix. (B) Illustration of the LF strip compartment and the LF strip schematic: the LF strip contains an extended sample pad that is positioned in a trough that prevents overflow of the LF sample pad and strip. (C) Schematic of the compartment with the NA-binding silica membrane; the inlet/outlet of the connected channels are indicated. (D) Schematic of the amplification microtube indicates how the lumen functions as both inlet and outlet. During amplification the opening of the lumen is well above the liquid surface. After amplification, HSLF buffer is added to tube through the lumen, and as a result the opening of the lumen will be below the liquid surface allowing it to function as an outlet.

Figure 4

The consecutive flow protocol. (a) Schematic for the analysis of the antibody path, the Test Line was a proprietary peptide mix (OraSure Technologies), and the Flow Line anti-human IgG. (b) Schematic showing consecutive flow applied to detect RT-PCR amplicons provided with a digoxigenin and biotin hapten as described by Corstjens et al. [8], which was developed to detect antibodies against infectious disease pathogens. For analysis of amplicons, the Test line was antidigoxigenin and the Flow Line digoxigenin.

Figure 5

On-chip RNA isolation protocol compared to a typical manual benchtop operation. The bench top RNA isolation protocol involves several centrifugation steps using spin columns provided with a silica purification membrane. For the chip protocol, these steps were replaced by on-chip vacuum filtration.

Figure 6

Optimization of the RT-PCR amplicon yield. (a) The effect of different wash buffers on the quality of on-chip RNA isolation was assessed by amplifying increasing amounts of CARD isolated RNA elute by RT-PCR. The volumes represent the amount of eluted RNA used in the amplification reaction using a 10 μL final assay volume. Note the decrease in amplicon yield with increased volume possibly due to the presence of residual EtOH. (b) Doubling of the primers and enzyme concentration and a 2°C lower annealing temperature increased the amplicon yield.

Figure 7

Analysis of saliva samples spiked with HIV RNA as Armored RNA and HIV antibodies on the dual path CARD. (a) Signals representing peak areas (emission in RFU after excitation with 980 nm IR light) of the Test and Flow Control lines. (b) Results are presented as Ratio Values calculated by dividing Test and Control line signals. Ratio values improve the interassay comparison obtained with different LF strips.